The Tn10-encoded Tet repressor (TetR) protein regulates the expression of tetracycline resistance genes in gram negative bacteria, e.g., Escherichia coli, in a tetracycline (Tc) dependent fashion (reviewed in Hillen & Berens, 1994). In the absence of Tc, a TetR protein dimer binds to operator sequences (tetO) and inhibits expression of the tetracycline resistance gene (tetA). When the inducer Tc enters the cell and binds to TetR, the affinity for tetO is reduced and TetR dissociates from tetO, allowing expression of tetA. The crystal structures of the TetR-[Mg—Tc]+ complex (Hinrichs et al., 1994; Kisker et al., 1995) and free TetR (Orth et al., 1998), and analysis of non-inducible TetR mutants (Müller et al., 1995), imply that the binding of Tc induces conformational changes in TetR. Dimerization of TetR is mediated by a four helix bundle, and residues which determine the dimerization specificity have been identified (Schnappinger et al, 1998). This has led to TetR based regulators which cannot heterodimerize.
TetR-based transcription activators have been developed which allow inducible expression of appropriately modified genes in a tetracycline dependent mode (Gossen & Bujard, 1992; Gossen et al, 1995) in various cellular systems of mammalian (Gossen & Bujard, 1992), plant (Weinmann et al, 1994; Zeidler et al, 1996) and amphibian (Camacho-Vanegas et al., 1998) origin, as well as in whole organisms including fungi (Gari et al., 1997), plants (Weinmann et al., 1994), Drosophila (Bello et al., 1998), mice (Kistner et al., 1996; Efrat et al., 1995; Ewald et al., 1996) and rats (Fishman et al., 1994; Harding et al., 1998).
Tetracycline controlled transactivators (tTA) are fusions between TetR and proper domains of transcriptional activators. In one such fusion protein, a major portion of the Herpes simplex virus protein 16 (VP16) was fused at the level of DNA to TetR. Yet, other tTA's demonstrate a graded transactivation potential resulting from connecting different combinations of minimal activation domains to the C-terminus of TetR (Baron et al., 1997). These chimeric “tetracycline controlled transactivators” (tTA) allow one to regulate the expression of genes placed downstream of minimal promoter-tetO fusions (Ptet). In absence of Tc Ptet is activated whereas in presence of the antibiotic activation of Ptet is prevented.
A “reverse tetracycline controlled transactivator” (rtTA) was developed which binds operator DNA only in the presence of some tetracycline derivatives such as doxycycline (Dox) or anhydrotetracycline (ATc), and thus activates Ptet upon addition of Dox (Gossen et al., 1995). Both tTA and rtTA are widely used to regulate gene expression in various systems (for review see Freundlieb et al., 1997).
Despite widespread use of Tet systems in academic and industrial research, as well as in some technical processes such as high throughput screening and fermentation, there are limitations which prevent their use in a number of areas because of the specific properties of the transactivators, and of the inducing effector substances. These limitations concern particularly:                the residual affinity of rtTA to tetO sequences in the absence of the inducer;        the relatively low susceptibility of rtTA towards Dox;        the interdependence between different domains of tTA and rtTA, that can affect the specificity of transactivator/operator interaction;        the stability of tTA and rtTA in different eukaryotic systems;        the relatively narrow temperature optimum of tTA/rtTA function;        the antibiotic activity of some of the best effector molecules; and        the restriction of effectors to substances of the tetracycline family.        
For example, the known rtTA described above has retained a residual affinity to tetO in the absence of doxycycline (Dox). This can lead to an intrinsic basal activity of rtTA responsive promoters, and indeed such increased basal levels of transcription have been observed in mammalian cell lines as well as in S. cerevisiae. Tc controlled expression using tTA and rtTA in S. cerevisiae has been published (Gallego et al., 1997; Gan et al., 1997; Belli et al., 1998a; Belli et al., 1998b; Nagahashi et al., 1998; Nakayama et al., 1998; Colomina et al., 1999). Gene regulation was achieved with tTA showing high expression of lacZ and low basal activities (Bari et al., 1997). In contrast, rtTA did not regulate expression in response to Tc due to extremely high basal expression, leaving no room for apparent induction of gene expression. Thus, an additional regulated repressor was introduced to lower the basal expression (Belli et al., 1998). Only this dual control system previously yielded reasonable induction factors in S. cerevisiae. In addition, the known rtTA is fully induced only at relatively high levels of Dox.
Moreover, it appears that active rtTA proteins cannot be synthesized in a number of systems including B-cells in transgenic (tg) mice, Drosophila melanogaster, and plants. Whether this is due to instabilities at the level of RNA or protein, or both is not entirely clear.
The known transactivators also exhibit a rather narrow temperature optimum. In mammalian systems, this does not pose a particular problem. By contrast, applying Tet regulation to plants will require an expanded temperature tolerance of transactivators.
Previously, the most efficient way of producing TetR variants was based on random or directed mutagenesis, followed by screening procedures that relied on TetR function in E. coli (Helbl & Hillen, 1998; Helbl et al., 1998; Müller et al., 1995; Hecht et al., 1993; Wissmann et al., 1991). TetR variants identified in this way were subsequently converted to tTA and/or rtTA fusion proteins whose properties were examined in eukaryotic systems. Frequently, the properties of TetR variants as identified in E. coli would not correlate with those of the corresponding tTA or rtTAs in eukaryotic cells. The main reasons for these inconsistencies are: (a) fusion of activator domains to TetR variants or introduction of mutations, e.g., mutations that confer the reverse phenotype, may negatively affect the overall function of the respective TetR variant; (b) the properties of tTA/rtTA's such as stability or the interaction with operator sequences is affected by differences in the cellular environment between E. coli and various eukaryotic systems; and (c) tetracycline and many of its derivatives are toxic in prokaryotes where they act primarily to inhibit protein biosynthesis, and thus limit screening procedures to sublethal concentrations of the effector molecule. By contrast, tetracyclines are tolerated at higher concentrations in eukaryotic cells.
It is therefore necessary to examine fully the useful sequence space of the Tet repressor. To this end, it is desirable to develop a screening method which is capable of rapidly and efficiently identifying novel variants of tTA and rtTA out of large pools of candidates produced by random, semi-random and directed mutagenesis.
Optimal application of tTA's and rtTA's in different eukaryotic systems will require the development of transactivators that are specifically adapted to defined tasks. Therefore, screening systems that are capable of identifying tTA/rtTA phenotypes directly in eukaryotes like yeast or other fungi will constitute a significant improvement over the current screening technology for the following reasons:                the phenotypes identified will directly reflect the properties of the transactivating fusion protein (TetR fused to an activation domain) in an eukaryotic system;        mutagenesis can be performed throughout the gene encoding the entire transactivator;        mutations within the activation domain can be included in the analysis; and        using yeast or other fungal systems will result in screening efficiencies that are comparable to those obtained in E. coli.         